We have found that Cyr61 is overexpressed in human breast cancers, and its forced expression in non-transformed breast cells results in robust tumor formation in nude mice. We hypothesize that high levels of Cyr61 produced and secreted by breast cancer cells, bind to integrin receptors to stimulate growth in an autocrine and paracrine fashion. Specific Aim 1: Correlate expression of Cyr61 in breast cancers with clinical parameters. Breast cancer tissue arrays will be stained for Cyr61, and results will be correlated with a variety of clinical information. We hypothesize that Cyr61 by binding to integrins activates the beta-catenin/TCFand Pi3KIAKT/mTORI4E-BPIlSK6 pathways. Replicate breast cancer arrays will be stained to determine if these downstream proteins are activated. Specific Aim 2: Determine pathways by which Cyr61 stimulates growth of breast cancer cells. Breast cell lines genetically engineered to have high Cyr61 expression, will be used to unravel Cyr61 signaling pathways employing a variety of techniques: dominant negative expression vectors, blocking chemicals, siRNA, anti-sense and purified Cyt61. Specific Aim 3: Investigate the effects of overexpression of Cyr61 on p53 function, we discovered that breast cells that overexpress Cyr61, have a non-functional p53. The mechanism of this extremely important finding will be explored. Specific Aim 4: Determine which domains of Cyr61 are necessary for enhancing the transforming ability of breast cancer cells. Cyr61 has well defined, conserved modules. Cyr deletional mutants will be stably placed into breast cells and examined for phenotypic changes. Specific Aim 5: Purify Cyr61 in order to test on breast cells and to develop an ELISA. Cyr61 will be purified and used to examine its effect on transformed and non-transformed breast cells. Also, we will make the first Cyr61 ELISA, which may have clinical and laboratory utility. Specific Aim 6: Determine the effects, in vivo of selective overexpression of Cyr61 in breast tissue. Transgenic mice that selectively overexpress wild type and deletionally mutant Cyr61 will be created to study the in vivo development of breast cancer. Specific Aim 7: Analyze the results of gene silencing of Cyr61 in breast cancer cells. Taking advantage of lentiviral and siRNA technologies, expression of Cyr61 will be inhibited in breast cancer lines that constitutively overexpress it; their phenotypic changes will be examined. Specific Aim 8: Study the therapeutic implications of overexpression of Cyr61 in breast cancers. Our preliminary observations that Cyr61 overexpressing breast cancer cells are chemo-resistant will be pursued. Also, to explore therapeutic possibilities, we will try to block the Cyr61 pro-growth/anti-apoptotic pathways in breast cancers using a variety of approaches. Taken together, our proposed studies will provide unique insights into the pivotal role that Cyr61 plays in breast cancer ant they may lead to novel therapeutic approaches.